CN109082415A - A kind of novel goose astrovirus Strain and its application - Google Patents
A kind of novel goose astrovirus Strain and its application Download PDFInfo
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Abstract
The present invention provides a kind of novel goose astrovirus for causing young goose gout, is 0122 plant of GD, and Wuhan University's China typical culture collection center is deposited on May 13rd, 2018, and deposit number is CCTCC No:V201820.Novel goose astrovirus provided by the present invention can be used for preparing vaccine or antibody for preventing young goose gout.The young goose gout vaccine safety of the virus that the present invention is screened, preparation is good, does not occur any locally and systemically adverse reaction as caused by vaccine.Analysis in storage life test Jing Guo character, safety testing, potency test data, indices are stable effective；Immune effect using serological method and Immunization method assessment vaccine has good commercialized development prospect the results show that the inactivated vaccine prepared in the present invention is capable of providing effective immunoprotection to gaggle.
Description
Technical field
The invention belongs to veterinary biologics technical fields, and in particular to a kind of novel goose astrovirus Strain and its answer
With.
Technical background
Gout is also known as uric acid mineralization, and nephrotic syndrome is to cause body since internal uric acid generates excessive or excretion disorder
Interior a large amount of uric acid accumulations, and joint, internal organs serosal surface and its hetero-organization interstitial are deposited in the form of lithate, to draw
It rises and suffers from fowl bradykinesia, arranges white loose stool, anorexia, failure, or even is dead, be the protein metabolism irregularity disease of Special Category
Disease.
Current goose gout occurs mainly in young goose, and 5~15 ages in days are multiple, sometimes visible 1 age in days young bird goose morbidity, a variety of kinds
Goose can fall ill, and the death rate is 30%~70%, and sick goose arranges lime sample loose stools within 2~3 days before the onset, and dehydration reduces, then jumpbogroup
There are the symptoms such as depressed spirit, material reducing, butt-swelling, dehydration, expiratory dyspnea.The visible pericardium of dissect is coated with by white lithate, is commonly called as
" cor villosum ", liver, air bag serosal surface and joint have a white uric acid mineralization, kidney enlargement, mottled, and ureterectasia is simultaneously
There is uric acid mineralization.
Cause the inducement of young goose gout very much, it is generally recognized that with forage protein too high levels, renal insufficiency, introduce a fine variety, raise
It is related to support mismanagement etc., but research recently is found after excluding these factors, gout still can occur, will die of illness goose dissect internal organs
Contact dyeing is carried out, does not find bacterium under the microscope after Gram's staining and Wright's staining.Scrape intestinal contents, physiological saline
Smear sets low a times microscopic observation, has no coccidian oocyst.It takes air bag and joint lime sample object to make smear, sets low a times microscopic observation,
It can be seen that the urate crystal object of a large amount of needle point sizes.According to raiser's description, clinical symptoms, dissect lesion, tentative diagnosis is doubtful
Cause like goose viral infection and destroy hepatic and renal function, leads to uric acid mineralization.
By the epidemiological survey to young goose gout, it is found that disease disease incidence in China gaggle becomes in increasingly rising
Gesture.Based on currently, not preventing and treating the commercialized vaccine (or antibody) of the disease both at home and abroad, which is also constantly spreading.It grinds
Safely and effectively vaccine and prevention and treatment are extremely urgent with Yolk antibody for system, it is necessary to overcome the problems, such as this by innovative technology.
Summary of the invention
The object of the present invention is to provide a kind of novel goose astrovirus for causing young goose gout symptom, and prepared by its strain
Kind of goose is immunized at inactivated vaccine, filial generation is made to obtain higher maternal antibody or Immune Laying Hens, collects high-immunity egg, extracts high-immunity egg
Middle IgY antibody, for preventing young goose gout, to make up the deficiencies in the prior art.
Novel 0122 plant of (Noval goose of goose astrovirus GD provided by the present invention for causing young goose gout symptom
Astrovirus Strain GD 0122), it is deposited in Wuhan University's China typical culture collection on May 13rd, 2018
The heart, deposit number are CCTCC No:V201820.
Novel goose astrovirus provided by the present invention can be used for preparing vaccine or antibody for preventing young goose gout；
The vaccine is inactivated vaccine.
The young goose gout vaccine safety of the virus that the present invention is screened, preparation is good, does not occur being drawn by vaccine immunity
Any locally and systemically adverse reaction risen.Pass through point of character, safety testing, potency test data in storage life test
Analysis, indices are stable effective；Using the immune effect of serological method and Immunization method assessment vaccine, the results show that
The inactivated vaccine prepared in the present invention is capable of providing effective immunoprotection to gaggle, has good commercialized development prospect.
Specific embodiment
The present invention is described in detail below with reference to embodiment.
The separation and identification of 0122 plant of 1 GD of embodiment
1, since in October, 2017, the young goose in the area such as Guangdong, Sichuan, Hubei occurs one for epidemiological survey
Kind of the death rate is higher, the frank communicable disease of gout.The disease is mainly in 5~15 age in days young bird geese, and various kind geese are easy
Sense carries out dissect to the young goose to die of illness, and main pathological change is as follows: pericardial thickening, has white crystals thing to adhere to or be coated with, custom
Claim " cor villosum ", liver is covered by lithate, and ureter is thicker, metabolism is obstructed, renal flares or pale enlargement, piebald kidney, directly
The accumulation of intestines end content white loose stool, gall-bladder and spleen enlargement, intestinal wall is thinning, serious case visible joint redness, joint
It is intracavitary to have uric acid mineralization.Single cases will appear serious enteron aisle embolism, similar to the typical disease symptom of gosling plague.Through facing
Bed investigation and laboratory testing, tentative diagnosis are novel goose astrovirus.Inventor's success is sent out from 10 ages in days of Guangdong goose field
A strain virus is isolated in sick gaggle.
2, virus purification chooses disease symptom and typically dies of illness Goose Liver, spleen, kidney, and the tissue such as pancreas 50~
100g is added in 1: 5 (w/v) ratio containing dual anti-(penicillin containing 1000U/ml+1000ug streptomysin/ml) with mortar grinder tissue
Sterile PBS (0.1mol/L, pH value 7.2) homogenate, 6000r/min is centrifuged 10 minutes after multigelation 3 times, and supernatant is taken to pass through
0.22um filter filtration sterilization saves backup after steriling test is qualified.Virus filtration liquid is inoculated with 12~13 age in days goose embryos, every embryo
0.2ml, 36~37 DEG C of incubations, every sunshine embryo 2 times discard goose embryo dead in 24 hours, harvest 24~168 hours dead goose embryos
With 168 hours survival goose embryo allantoic liquids, 2~8 DEG C of coolings 12~24 hours are set, harvest goose embryo allantoic liquid.
3, viral identification
3.1 coagulation identifications difference aseptic collection SPF chickens and goose blood 5ml, according to 2015 editions " Chinese veterinary pharmacopoeia " methods
It is prepared into the red cell suspension of 0.8%, 1% and 2% concentration respectively, 4 DEG C save backup.The goose embryo virus liquid of harvest is subjected to HA
Whether titration, detection isolated strain have the characteristic for being aggregated these red blood cells.As a result: separation poison cannot be aggregated SPF chicken and
Goose hematid can not be allowed to be aggregated even if changing the concentration of red cell suspension, and red blood cell control is set up.
3.2 physicochemical properties examine virus liquid respectively through chloroform, ether, hydrochloric acid (pH3), (50 DEG C, 1 hour) of temperature processing
Afterwards, 12 age in days goose embryos (0.2ml/ embryo) are inoculated with, separately set physiological saline processing group as control.37 DEG C are cultivated 7.As a result: separation
The strain of poisons ether, chloroform and hydrochloric acid (pH3) processing, does not influence the proliferation in viral goose embryo, and dead, PCR inspection occurs in goose embryo
It is positive to survey result, shows that virus does not have lipid cyst membrane, has resistance to ether and chloroform, it is acidproof.Virus stablizes heat treatment, energy
Resist 50 DEG C, 1 hour.The nucleic acid type of isolated strain is RNA.
3.3 PCR identification takes separation strain virus 200ul, extracts viral RNA using RNA extracts kit, DNA extracts examination
Agent box extracts viral DNA, and after RNA reverse transcription is at cDNA, PCR detects that goose pair is viscous, H5, H7, H9 subtype avian influenza, reovirus
And astrovirus, DNA PCR detect circovirus, aviadenovirus, gosling plague, Muscovy duck parvovirus.1% agar after PCR
Result is observed in sugared gel electrophoresis.
PCR reaction system: total volume 25ul
It is sequentially added in 0.5m1 PCR pipe
10×Ex Taq Mix-----------------12.5u1
Upstream primer (20pmol) --- --- --- --- --- 1u1
Downstream primer (20pmol) --- --- --- --- --- 1u1
DNA----------------------------2ul
Total volume 25u1 is complemented to sterile deionized water 8.5ul, PCR reaction carries out in PTC-100 gene-amplificative instrament.
PCR response parameter: initial denaturation condition is 94 DEG C, and 3 minutes, Denaturing was 94 DEG C, and 30 seconds, annealing temperature and time were 50 DEG C,
30 seconds, elongating temperature was 72 DEG C and extends 45 seconds that 35 circulations, last 72 DEG C extend 10 minutes.
Through expanding, isolated strain has only amplified the corresponding target fragment of astrovirus, with expected purpose clip size phase
Symbol.Goose pair is viscous, H5, H7, H9 subtype avian influenza, reovirus, circovirus, adenovirus, Goose Parvovirus, the tiny disease of kind duck
The detections such as poison are feminine gender.After the target fragment of amplification is sequenced, by NCBI BLAST compare analyze, separation poison with
The astrovirus sequence homology that NCBI is announced is closest, but there is also biggish difference, homology only has 60% or so, shows
The virus screened is a kind of novel virus.
3.4 pathogenicity
3.4.1 7 ages in days are inoculated with intravenous injection and chest muscle approach respectively by 0122 plant of GD to the pathogenic of chicken
SPF chicken each 5,1.0ml/ only, is observed 14, and SPF chicken is strong to live, and without any clinical manifestation, is attacked dissect on the 14th after poison, is had no and appoint
What lesion.
3.4.2 2 ages in days young bird is inoculated with intravenous injection and chest muscle approach respectively by 0122 plant of GD to the pathogenic of duck
Duck, 1.0ml/ only, are observed 14, and duckling is strong to live, and without any clinical manifestation, are attacked dissect on the 14th after poison, are had no any lesion.
3.4.3 2 age in days young bird geese 10 are inoculated with intramuscular injection path by 0122 plant of GD to the pathogenic of young goose,
1.0ml/ only, starts to occur dead for young goose the 4th day after attacking poison, and peak mortality concentrates on attacking after poison 7~9, and the death rate is reachable
To 8/10, the young goose that falls ill arranges white loose stool, and dissect is died of illness young goose, it is seen that kidney whitens or mottled, ureter enlargement, liver throughout
There is cheesy uric acid mineralization in joint in white point, the visible pericardium apurinic acid mineralization of serious person.
3.5 viral immunogenics, which are examined, has carried out Study On Immunogenicity for the virus liquid after subculture is purified.The strain system
Adult female kind goose is immunized at inactivated vaccine, immune rear institute's produce surviving of son generation at 2 monthly age, which can generate, completely attacks malicious protection, illustrates this
Strain is one plant of ideal antigen.
Other popular strains that 3.6 virus-specifics and neutralization examine the strain and the same period to separate are through subculture and pure
Inactivated vaccine is made in change, and 30 age in days SPF chickens are immunized respectively, and head exempts from dosage 0.5ml/ only, and booster immunization dosage is 1.0ml/, often
It is immune primary every 2 weeks, be immunized 3 times altogether, 3 exempt from after blood was collected within 10 days, separate serum, it is specific through 56 DEG C of inactivations progress in 1 hour
Test.
10 times of 0122 strain virus liquid of GD are diluted, is mixed in equal volume with the positive serum of the strain and epidemic strain respectively, 37
DEG C neutralize 1 hour after, be inoculated with 12 age in days goose embryos, 0.2ml/ pieces, observed 168 hours after inoculation.As a result, it has been found that the positive of the virus
In serum and group goose embryo is all strong lives, in the positive serum of other epidemic strains and group only 70%~80% it is strong live, in non-increase serum
The virus group goose embryo of sum is all dead, and the positive serum for illustrating that the strain that the present invention screens is prepared as antigen screens the present invention
The neutralising capacity of virus be significantly better than the positive serum of popular strain preparation.
Embodiment 2
The preparation of 0122 plant of seed culture of viruses of GD
0122 plant of GD, 10 times of seed culture of viruses is diluted, 12~13 age in days health goose embryos are inoculated with, 0.2ml/ embryo sets 60% humidity, 37
Goose embryo dead in culture 168 hours, 24 hours discards in DEG C incubator, and dead goose embryo takes out immediately within 24~168 hours, sets 4
It DEG C saves, until 168 hours, embryo living is all taken out, 4 DEG C after storage 12~24 hours, are collected goose embryo allantoic liquid.Method according to this
The goose embryo allantoic liquid of above-mentioned harvest is continued into 5 generation of subculture in 12~13 age in days goose embryos, is respectively labeled as D2~D6 generation.Measurement is every
Generation viral level.Sterile and viral level >=10 will be examined5.0TCID50Virus liquid mixing, quantitative separating, freeze-drying save.
Embodiment 3
The preparation of 0122 plant of antigen of GD
1, inoculation takes 0122 plant of seed culture of viruses of GD, makees 10 times of dilutions, inoculation 12 with sterilizing PBS (0.1mol/L, pH value 7.2)
~13 age in days health goose embryos, 0.2ml/ embryo seal pin hole after inoculation, set 36~37 DEG C and continue to be incubated for, it is not necessary to turn over embryo.
2, after being incubated for and observing goose embryonic breeding kind, every sunshine embryo 2 times discards goose embryo dead before 24 hours.Until 168
Hour, no matter it is dead whether, all take out, gas chamber is upright upwards, is placed in 2~8 DEG C of coolings 12~24 hours.
3, it harvests and takes out cooling goose embryo, with iodine tincture disinfection gas chamber position, gas chamber portion ovum is then stripped with aseptic procedure
Shell throws off egg membrane, breaks chorioallantoic membrane and amnion (guarding against yolk rupture), draws blastochyle.To each goose before drawing blastochyle
Embryo is gone through, and all fetus corruption, blastochyle is muddy and has any pollution suspicious person, should be discarded.Dead germ and embryo living harvest respectively, often
Several goose embryos are divided into one group, draw blastochyle and are put into the container of same sterilizing, set -20 DEG C of preservations.
Embodiment 4
The preparation of inactivation and the inspection of semifinished product and vaccine of 0122 strain virus liquid of GD
1 inactivation imports 0122 strain virus liquid of GD in inactivation bottle, metered 10% formalin, opens blender and stirs
It mixes, mixes them thoroughly, the ultimate density of formaldehyde is 0.1%.It is imported in another inactivation bottle after adding formalin, to avoid bottleneck
The virus nearby adhered to fails to contact inactivator.4 DEG C inactivate 36 hours, put 2~8 DEG C of preservations if any residue, should be no more than 1
Month.
2 inspection of semifinished product
2.1 steriling tests take the virus liquid of inactivation, test by version " Chinese veterinary pharmacopoeia " annex in 2015, sterile life
It is long.
2.2 inactivations are examined the virus liquid after inactivation, and 12~13 5 pieces of age in days goose embryos, 0.2ml/ embryo, every sunshine embryo 2 are inoculated with
It is secondary, it observes 7, harvests goose embryo allantoic liquid, PCR detection is negative, and then blind passage is primary again by blastochyle, observes 7, PCR is detected still
When unnegative, it is believed that inactivation is complete.
Seed culture of viruses is carried out 10 times with sterilizing PBS (0.1mol/L, pH value 7.2) and is serially diluted by the measurement of 2.3 viral levels, is taken
10- 3、10- 4、10- 5、10- 64 dilutions are inoculated in 12~13 age in days goose embryos respectively, and each dilution is inoculated with 5 pieces, every embryo
0.2ml.Virus positive control and PBS negative control are set simultaneously, set 60% humidity, 37 DEG C of incubator cultures and is observed 168 hours,
Goose embryo embryo PCR dead or living detects positive and is judged to infect, and calculates EID50。
The preparation of 3 inactivated vaccines
It takes the antigen liquid of inactivation to be emulsified with SEPPIC ISA71 oil adjuvant by proper proportion, first starts motor low speed and stir
Mix ISA71 adjuvant 2 minutes, at the same slowly be added antigen liquid, 10000r/min, emulsify 15 minutes, oil emulsion inactivated vaccine.
4 packing quantitative separatings, seal, and adhesive label, set 2~8 DEG C of preservations.
Embodiment 5
Vaccine product inspection
1 character
Appearance milky white emulsion.
Dosage form water-in-oil type.A cleaning suction pipe is taken, a small amount of vaccine is drawn and instills in cold water, in addition to the 1st drop, answers indiffusion.
Stability is drawn vaccine 10ml and is added in centrifuge tube, with 3000r/min centrifugation 15 minutes, the water phase that tube bottom is precipitated
0.5ml should be no more than.
Viscosity is carried out by existing " Chinese veterinary pharmacopoeia " annex, should meet regulation.
2 loading quantity inspections are checked by existing " Chinese veterinary pharmacopoeia " annex, should meet regulation.
3 steriling tests are tested by existing " Chinese veterinary pharmacopoeia " annex, answer asepsis growth.
4 safety verification subcutaneous inoculation, 1 age in days young bird goose 10, the subcutaneous branch of every neck vaccinate 1.0ml, observation 21
Day, growth and development is normal, and the state of mind is good, and dissect injection site vaccine absorbs good, the inflammation such as no redness, tissue necrosis
Reaction.
The detection of 5 immune effect of vaccine
Vaccine is subcutaneously or intramuscularly injected kind goose 10 with the dosage neck of 2.0ml/ only by 5.1 serological methods, is separately set
Not immunized controls 10.28 days after immune, immune goose and control goose are taken a blood sample respectively, are separated serum, are used agar immunodiffusion test
Method measures serum antibody titer, reporter antibody result.
Vaccine is subcutaneously or intramuscularly injected kind of a goose with the dosage neck of 2.0ml/ only by 5.2 Immunization methods, is separately set and is not immunized
It compares.1~2 month after immune, hatching egg is collected, hatching and nestling goose only carries out attacking poison, while setting and not being immunized to 1 age in days young bird goose 10
Control 1 age in days young bird goose 10 of filial generation is only used as compareing.All immune geese and control goose filial generation, intramuscular injection virus liquid 0.5ml/ is only
(viral level 105.2EID50/0.2ml).It observes and records immune goose in detail and compare the incidence of goose filial generation.
As the result is shown: after 3 batches of vaccine immunity kind geese, 28 days after exempting from, fine jade, which expands antibody titer, can reach 8/10 with Shangyang
Property, and compare and goose is not immunized, it is feminine gender that fine jade, which expands antibody titer,；1 age in days young bird goose maternal antibody of filial generation is high, attacks 3 batches of vaccines after poison
Immune goose filial generation is observed 14, and protection number is no less than 8；Control goose attacks malicious all morbidities, and dead 6, dead goose is cutd open
Inspection, visible kidney enlargement are whitened or mottled, the typical cases such as serious appearance " cor villosum " and the cheesy uric acid mineralization of joint
Lesion.It is detailed in the following table 1.
13 batches of vaccine potency inspection results of table
To sum up, the present invention is a kind of inactivation that immune effect is more satisfactory using the vaccine of 0122 plant of GD preparation of screening
Seedling promotes the maternal antibody of filial generation, can effectively prevent the generation of filial generation young bird goose gout after kind of goose is immunized.
Claims (6)
1. a kind of novel goose astrovirus, which is characterized in that the deposit number of the novel goose astrovirus is CCTCC No:
V201820。
2. novel goose astrovirus described in claim 1 is preparing the application in vaccine.
3. application as claimed in claim 2, which is characterized in that the vaccine is inactivated vaccine.
4. application as claimed in claim 3, which is characterized in that the vaccine is for preventing young goose gout.
5. novel goose astrovirus described in claim 1 is preparing the application in antibody.
6. application as claimed in claim 5, which is characterized in that the antibody is for preventing young goose gout.
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| CN112280750A (en) * | 2020-10-22 | 2021-01-29 | 山东农业大学 | Novel goose astrovirus with cross-species transmission capability and application thereof |
| CN112341539A (en) * | 2020-10-22 | 2021-02-09 | 山东农业大学 | Yolk antibody for preventing and treating novel goose astrovirus with cross-species transmission capability and preparation method thereof |
| CN112999343A (en) * | 2021-03-23 | 2021-06-22 | 北京世华康源生物科技有限公司 | Inactivated vaccine of goose astrovirus and preparation method thereof |
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